Mass spectrometers are commonly used for the determination of the mass of analyte molecules. In these instruments, ionized molecules are typically either created or introduced into a high vacuum chamber and accelerated to a known kenetic energy. Magnetic fields and electric fields are then used in various methods and fashions for mass selection, mass filtering, and thereby mass determination of the ionized molecules. Among the various types of mass spectrometers commercially available today, there are included magnetic sector, time-of-flight (TOF), ion trap, quadrupole, and ion cyclotron resonance instruments. There are also available instruments that are combinations of the various techniques of mass analysis.
In a typical sector mass spectrometer, a magnetic field (magnetic sector) mass analyzer is scanned over a mass range of interest causing an ion beam output spectrum of mass versus magnetic field intensity. There is commonly also an electrostatic analyzer (ESA) either before or after the magnetic sector, so as to select only ions of a narrow energy distribution, thereby improving resolution, a measure of the selectivity of the mass analysis. Scanning of the magnetic fields and electric fields is a relatively slow process resulting in low efficiency as the ionization is typically, although not always, a continuous process.
By contrast, in a typical time-of-flight mass spectrometer, the entire mass range is analyzed in a single experiment, limited in time only by the mass dependant flight time of the ions in the vacuum chamber, a period measured in microseconds. Time-of-flight instruments have a significant duty cycle advantage over scanning instruments which require a much longer time period to scan the selected mass range.
In mass spectrometry, it is desirable not only to investigate the mass of the intact analyte molecules, but to also be able to dissociate selected analyte molecules (precursor ions) and investigate the mass of the dissociated product ions (fragment ions), and thereby investigate the structure of the precursor analyte molecules. In a typical mass spectrometer designed for MS/MS experiments, there is an MS1 mass analyzer wherein the analyte precursor molecule is mass analyzed and selected, a dissociation region wherein the mass selected precursor ion is collided with a gas, photons, or a surface, thereby causing dissociation of the precursor ions, and an MS2 mass analyzer wherein the resulting product ions are mass analyzed. This is commonly referred to as MS/MS spectrometry or tandem mass spectrometry. Tandem mass spectrometry plays an essential role in the structural analysis of a wide variety of compounds including biomolecules, such as peptides, proteins, and oligonucleotides.
In Collision Induced Dissociation (CID), the mass selected precursor ions from MS1 are passed through a region of relatively high pressure, causing the precursor ions to collide with a target gas molecule. The energy imparted to the precursor ion in such a collision will frequently lead to dissociation of the precursor ion. The efficiency of the CID process is determined in great part on the choice of target gas and the density of the target gas in the collision cell and is proportional to the kenetic energy (KE) of the precursor ion.
The most common MS/MS instruments have until recently been high performance tandem sector instruments. These instruments tend to be large and expensive, and, due to the scanning nature of sector instruments, the product ion collection efficiency has been very low.
An alternative solution is the use of a TOF analyzer as a second stage (MS2) for a sector instrument. Clayton and Bateman (Rapid Communications in Mass Spectrometry (RCM) 6 (1992) 719) proposed such an instrument that employed orthogonal extraction into a TOF analyzer. However, to perform high energy CID experiments, only an xe2x80x9cin-linexe2x80x9d arrangement can be considered. An in-line arrangement also provides higher CID ion collection efficiency.
An in-line tandem TOF system was proposed by Davis and Evans in U.S. Pat. No. 5,180,914. In their system, a quadrupole field, pulsed, ion storage device was used to decelerate and mass analyze ions in a TOF MS1. The ions then passed through a dissociation region of a few millimeters where a timed laser pulse was applied (Photo Dissociation Spectrometry), after which the fragment ions (as well as the remaining parent ions) entered a TOF MS2 where the product ions were mass analyzed using a quadrupole field reflectron.
A reflectron (or ion mirror) as disclosed, for example, in Mamyrin et al. U.S. Pat. No. 4,072,862, is an electric field device that reflects ions backwards so as to increase the ion flight times and thereby increase the temporal resolution of the spectral results. Ion mirrors have the ability to correct the kinetic energy (KE) differences of ions of the same mass, thereby improving the quality of the mass spectrum. A true parabolic field reflectron is known to be energy independent for ions of the same mass over a very large mass range (Davis et al. U.S. Pat. No. 5,077,472), and has a single spatial focus point for ions irregardless of mass. This type of reflectron can correct for very large KE differences in the temporal focusing of ions. A disadvantage in using a parabolic field reflectron is that the spatial focal point of such a reflectron is located exactly at the entrance to the reflectron. In this invention, an offset parabolic field reflectron is introduced. Use of an offset parabolic field moves the reflectron spatial focal point beyond the entrance of the reflectron, thereby providing for field free regions to exist between the reflectron and its focal point.
Two groups have proposed in-line sector and TOF combinations: Derrick et al. (Int. J. Mass Spec. Ion Proc.) constructed a system based on some of the principles of the tandem TOF design of the Davis and Evans patent. In their implementation, a linear field two-plate ion buncher and a quadratic field planar symmetry reflectron were indicated. A parabolic curve of the shape V=Kd2 is independent of energy variations, however, there is no field free drift region allowed prior to the focus. In a parabolic field reflectron, the spatial focal point of the reflectron will be located exactly at the entrance to the reflectron.
Cotter, Cornish, and Musselman (RCM 8 (1994) 339) proposed the use of a curved field reflectron in a tandem sector/TOF instrument, however, a method of selection and focusing of the analyte precursor ions was not considered. In a curved field reflectron, field free regions may be defined in front of the reflectron. In TOF systems, these field free regions are commonly referred to as L1 and L2. In a curved field reflectron, ion flight times for a given mass are not completely energy independent. In an offset parabolic field reflectron, above a low energy threshold determined by the offset value, ion flight times of a given mass are completely energy independent, an important feature of this invention. In this invention, an offset parabolic field reflectron is used to achieve very high mass accuracy and resolution over the entire MS2 product ion mass range, above a low energy threshold determined by the offset value.
It will be recognized by those skilled in the art that other types of mass spectrometers, e.g., MALDI-TOF (Matrix Assisted Laser Desorption), or ESI-TOF (Electro Spray Ionization) instruments could, and have been, substituted for the sector instrument as MS1. The invention described herein has application in these other types of mass spectrometers, as well as the traditional sector instruments.
Briefly, according to this invention, there is provided a tandem mass spectrometry method with collision induced dissociation (CID) comprising the steps for: a) using a first mass spectrometer to select precursor ions of a selected mass, b) forming a packet of precursor ions, c) assigning a focusing energy to each packet of precursor ions in the ion buncher so as to bring the ions into temporal focus at some point in space, d) fragmenting the selected precursor ions near the spatial focus point to form product ions, e) passing the precursor and product ions into an offset parabolic field ion mirror (reflectron) for providing TOF dispersion among product ions of differing mass-to-charge ratios while maintaining near zero flight time dispersion (at the focal point) among product ions of the same mass-to-charge ratios but having large energy differences, and f) detecting the arrival times of the precursor and product ions. Preferably, the precursor and product ions pass through a field free region.
Preferably, the packets of precursor ions are formed by assigning a focusing energy pulse to eject ions from an ion source region in MS1 or gating a pulse of near mono-energetic precursor ions to an ion buncher.
Also, according to this invention, there is provided a tandem mass spectrometry apparatus comprising: a) a first mass spectrometer for selecting precursor ions of a predefined mass, b) apparatus for forming ion packets, preferably, a device for assigning a focusing energy pulse in the ion source, or an ion gate for forming a packet of precursor ions, alternatively, an ion buncher for applying a focusing pulse, c) a collision chamber for fragmenting the bunched precursor ions near the spatial focus point so as to form product ions, d) an ion mirror (reflectron) for providing TOF dispersion among product ions of differing mass-to-charge ratios while maintaining near zero flight time dispersion at the detector among product ions of the same mass-to-charge ratio and having large energy differences, and e) a detector for detecting the arrival times of, the precursor and product ions.
Preferably, an ion buncher is provided for spatially focusing a mono-energetic pulse of precursor ions. Preferably, a field free region is provided through which the precursor and product ions pass. Preferably, the bunching device is capable of precisely focusing relatively long ion beam pulses or clusters, thereby increasing the duty cycle, and therefore sensitivity, of a measured signal. This requires special means for providing velocity compensation across the ion path region. This type of buncher is referred to herein as a long buncher.
Preferably, the ion mirror is provided with a uniquely-shaped voltage distribution which permits significant flight time dispersion between product ions of differing mass-to-charge ratios while maintaining near zero flight time dispersion among fragments of the same mass-to-charge ratios having large energy differences. This energy independence is necessary due to the large energy distribution imparted by the focusing pulse to the precursor ions. The voltage distribution in the ion mirror that provides this property, and a focus point in a field free region outside of the ion mirror, is a parabolic function that is offset from the origin. The voltage can be described by the following equation: V=V0+K(dxe2x88x92d0)2.
Ions generated in an ESI-TOF or MALDI-TOF source may be analyzed in a similar fashion without the use of a an ion buncher where the focusing voltage pulse is applied to the precursor ions in the source.